Reduction or elimination of microorganisms on surfaces is important in a broad variety of applications. One approach to interfere with the ability of microorganisms to survive on various materials is to modify the surface of those materials by attachment of antimicrobial agents.
Deciding how best to attach an antimicrobial agent to a material is guided, at least in part, by the planned end-use of the material. One important and useful consideration is that the antimicrobial activity be persistent. This may be achieved by permanently attaching the antimicrobial agent to the surface, so that it is unable to migrate or leach away from the modified material surface when the modified material is exposed to fluids. For example, for applications in which the modified material will come into contact with aqueous fluids, it is important that the antimicrobial agent is not rinsed away when the modified material comes into contact with aqueous fluids. For applications in which the modified material will come into contact with aqueous biological fluids, it is important that the antimicrobial agent is not rinsed away, or otherwise inactivated, when the modified material is exposed to aqueous biological fluids. For applications in which the modified material is to be used repeatedly, it is important that the antimicrobial agent is not washed or rinsed away when the modified material is washed or rinsed in fluids in between repeated uses. An additional consideration in the development of approaches intended to meet the needs of these and related applications, is that some microorganisms have been found to possess the ability to develop resistance to certain antimicrobial agents, such as antibiotics or silver. Currently available approaches do not adequately address all of these considerations.
By their design, approaches utilizing leachable active agents (such as triclosan, silver compounds, or biguanides) to impart antimicrobial activity to materials suffer from eventual depletion or loss of the antimicrobial activity conveyed by the leachable active agents. Depletion or loss of antimicrobial activity can occur especially when the materials come into incidental contact with fluids, or during intentional contact with fluids during washing or rinsing procedures employed between repeated uses of the modified material. In addition, the leaching of certain active agents may prohibit or limit the use of these approaches in applications where the leaching of the active agent will cause undesired consequences (e.g. leaching into open wounds, leaching onto products intended for human consumption, and staining of skin).
One example of an approach utilizing a potentially leachable active agent is Burba et al. (U.S. Pat. No. 5,154,932), which discloses a method for providing antimicrobial activity to a formulation or product which have negative surface charges, effective for deactivating microorganisms, said method comprising adding to the formulation or product an amount of a positively charged layered crystalline mixed metal hydroxide sufficient to impart antimicrobial activity to the formulation or product.
Another example of an approach utilizing a potentially leachable active agent is Lyon et al. (U.S. Pat. No. 6,042,877), which discloses a method of making an antimicrobial article comprising providing a substrate, forming a solution comprising a chelating polymer and a metal ion, depositing the solution on the substrate, drying the substrate to form a coated substrate, and adding a potentiator to the coated substrate to form the antimicrobial article.
Other approaches have employed methods that attach silane-based quaternary ammonium compounds to particular substrates via a siloxane bond. For example, AEGIS Environments' product line includes products that utilize polymers of 3-(trimethoxysilyl)propyldimethyl octadecyl ammonium chloride. According to product literature, AEM 5700 is 43% 3-(trimethoxysilyl)propyldimethyloctadecyl ammonium chloride in methanol, which can be used to coat the surface of textiles. This is not a polymeric compound; although, some interlinking of the applied silane may occur after application to the substrate. These types of materials tend to impart a hydrophobic character to the substrates, and are thus less than suitable for many applications. Also, the hydrolytic instability caused by the bulky (C18 quat) substituent on the siloxane tends to make the materials prone to lose their antimicrobial activity.
Another example utilizing silane-based quaternary ammonium compounds is Blank et al. (U.S. Pat. No. 5,035,892), which discloses a method of inhibiting the proliferation of potentially destructive microorganisms on a substrate comprising treating the substrate with an effective amount of an antimicrobial superabsorbent composition formed of a crosslinked hydrophilic sodium salt form of a partially neutralized acrylic acid based polymer gel, the polymer gel having covalently bonded thereto an organosilane, the organosilane being present in an amount to prevent hydrophobing and reduction of the absorbent capacity of the polymer gel. Unfortunately, in practice, the amount of organosilane must often be reduced to below a desirable antimicrobially efficacious level in order to prevent the aforementioned hydrophobing. Another example is Blank (U.S. Pat. No. 4,847,088), which discloses a method of inhibiting the proliferation of potentially destructive microorganisms on a substrate comprising treating the substrate with an effective amount of the synergistic antimicrobial composition comprising a mixture of (a) an organosilane; and (b) an acid. Again, this example uses siloxanes as described above.
The silane-based quaternary ammonium systems suffer from other drawbacks as well. The long alkyl chains used (typically C-18) tend to make the treated material hydrophobic—not a desirable property for an absorbent material such as a wound dressing. Additionally, it has been found that the silane-quats are deactivated in the presence of blood or other proteinaceous material (see EP 0136900).
Another example is Shiau et al. (U.S. 010043938 A1), which discloses a process for producing an antimicrobial article comprising (a) dissolving a predetermined amount of quaternary ammonium organosiloxane salt in water; to make a solution of about 0.05 to 20 wt. % of the salt, (b) mixing a ground calcining aid with a solvent to make a solution, wherein said calcining aid to solvent ratio is from 1:1 to 1:10, (c) soaking a preformed honeycomb-shaped substrate into the solution of the aforesaid step b; followed by drying and calcining at 400-1500° C., (d) impregnating the calcined honeycomb-shaped substrate with the salt solution of the aforesaid steps a and e, drying the impregnated substrate at 50° to 200° C. These excessive temperatures (400° to 1500° C.) are clearly unsuitable for common substrates such as cellulose and polymers.
These shortcomings have been overcome, in part, by Batich in U.S. Pat. No. 7,045,673 and application U.S. 020177828 A1. The materials and methods described in those references teach that an antimicrobial material can be prepared by graft polymerization of an antimicrobial quaternary ammonium monomer onto a substrate such as cellulose. Still, that method has limitations and shortcomings. Namely, it requires a vinyl polymerization step which must be conducted under an oxygen-free atmosphere, which is a costly restriction to commercial development. Furthermore, the process described therein is wasteful in terms of utilization of antimicrobial cationic polyelectrolyte (for two reasons). First, a large excess of polymerizable quaternary ammonium compound must be utilized, and most of that is not incorporated into the final product. Second, the required grafting levels (up to more than 40% antimicrobial “add-on”) are at least an order of magnitude greater than is required by the present invention, in order to obtain the same level of antimicrobial efficacy. These shortcomings are discussed further elsewhere in the current application.
Lin describes (“Mechanism of Bactericidal and Fungicidal Activities of Textiles Covalently Modified with Alkylated Polyethyleneimine” Biotechnology and Bioengineering v83(2), p 168-172, Jul. 20, 2003) a process for covalently attaching hydrophobic polycations to woven textiles using a six-step process that requires various organic solvents and long processing times. The observed microbicidal activity indicated a range of 88% to 99% reduction for various organisms. The process described by the current application is shorter, faster, safer, and more economical than the process described by Lin. In addition, the material produced by the method of the current invention is orders of magnitude more effective in reducing the microbial activity, and is not hydrophobic. The shortcomings of the method described by Lin are cited in the following reference: Daewon Park, Jun Wang, and Alexander M. Klibanov, “One-Step, Painting-Like Coating Procedures To Make Surfaces Highly and Permanently Bactericidal” Biotechnol. Prog. 22, p 584-589 (2006). A similar process was used by Lee (Biomacromolecules 5 p877-883 (2004)).
Abel (“Preparation and investigation of antibacterial carbohydrate-based surfaces” Carbohydrate Research 337 (2002) p 2495-2499) describes the covalent attachment of low molecular weight (non-polymeric) quaternary ammonium moieties to cellulose substrates. Like the methods of Lin and Lee, Abel's method is a multistep process which requires the use of hazardous and/or flammable organic reagents and solvents such as p-toluenesulfonvlchloride, pyridine, and acetonitrile.
BacStop™ is sold as a fabric sanitizer by Edmar Chemical Company (Cleveland, Ohio). The product contains 50% didecyldimethylammonium chloride (DDDMAC). It is designed to be added to the final rinse cycle of a laundering process. BacStop™ is claimed to reduce bacterial count by 99.9% (3-log reduction) for Staph. aureus and Klebsiella pneumoniae, and to impart a residual bacteriostatic finish to fabrics. Such a residual effect is not unexpected, in that several rinses with fresh water would be needed for full removal of just about any soluble chemical from a fabric to which it has been applied. The active ingredient (DDDMAC) is a non-polymeric quaternary ammonium compound. The BacStop™ product label does not claim a residual antimicrobial property for fabrics that have been treated with the product—only a residual bacteriostatic property is claimed. It is demonstrated in comparative examples presented below that monomeric quaternary ammonium compounds such as DDDMAC are not effective in the practice of the current invention, as they do not produce a non-leaching bond with a substrate to give an inherently antimicrobial material with a desirable degree of antimicrobial efficacy.
Senka, in JP 09078785 describes some of the shortcomings of silane-based quaternary ammonium antimicrobials for the preparation of inherently antimicrobial materials (such as those described by Blank—see above). Senka teaches that an antimicrobial coating can be formed on non-celluosic substrates using a copolymer of a quaternary ammonium compound and acrylic acid derivatives. The copolymer has the property that it is soluble in aqueous solution; however, once the solution is dried, the resulting dried solid polymer becomes insoluble. Such behavior can be described as “self-crosslinking” in that the polymer (or copolymer) spontaneously crosslinks upon drying. Thus, a solution of the copolymer can be applied to a substrate and after drying an insoluble coating with antimicrobial properties is produced. Because the formation of the coating does not depend on interactions between the substrate and the coating, it is possible to apply this coating to inert substrates such as synthetic polymers. One skilled in the art would realize that these coatings are expected to be flexible when wet, or sufficiently hydrated; however, they would be expected to be stiff and brittle when dried. This is likely to cause undesirable changes in the physical properties of the material, such as stiffness, feel or hand. It is also likely that distortion, stretching, or folding of the underlying substrate would likely cause the dry coating to fall apart and detach from the substrate, particularly since there is no specific bonding between the coating and substrate. The dried copolymer presumably has become crosslinked due to interaction of the positively-charged quaternary components and the acrylic acid derived components, which are expected to be negatively-charged at neutral pH. Interaction of the oppositely-charged components of the copolymer is also likely to cause some screening of the positive charge provided by the quaternary component, and resulting in a reduction of antimicrobial efficacy. Furthermore, the simple fact that the coating consists of another component in addition to the quaternary component necessarily dilutes the charge density provided by the quaternary component, and thus also will reduce the ultimate antimicrobial effect compared to a polymer that consists of 100% quaternary component. These shortcomings are discussed further below.
In general, coatings are not a desirable approach to modification of cellulosic substrates such as textiles and wound dressings. While some coatings may form a very strong bond with a substrate, the attractive forces responsible for a robust and useful coating are generally between components of the coating (within the coating itself) rather than between the coating and the substrate. Coatings are generally somewhat thick (like paints, for instance), and can drastically affect the surface properties of textiles or other substrates. This can happen, for instance, by blocking or filling-in of porosity, or cementing-together of individual fibers. Additionally, some coatings require a curing step after application, in order to prevent subsequent dissolution.
Sawan (U.S. Pat. No. 6,264,936) describes an antimicrobial material which can be used to form on the surface of a substrate an antimicrobial coating or layer which kills microorganisms on contact. The antimicrobial coating or layer, characterized in the reference as “non-leaching,” is a combination of an organic matrix immobilized on the surface of the substrate to having biocidal metallic materials associated with the matrix. When a microorganism contacts the coating or layer, the biocidal metallic material is transferred to the microorganism in amounts sufficient to kill it. Specifically, the metallic antimicrobial agent used is silver. Although this method purports to provide a “non-leachable” coating, the mere fact that the metallic antimicrobial agent “is transferred” to the microorganism is contrary to the common definition of non-leachable. Furthermore, it is known that although silver and silver salts have very low solubility, the mechanism of antimicrobial activity is dependent on a finite solution concentration of silver ions. Indeed, Sawan later (column 3, line 9) qualifies the above statement to read “substantially low leachables”. In a preferred embodiment of Sawan's patent, the organic material comprises a polyhexamethylene biguanide polymer which is crosslinked with an epoxide, such as N,N-bismethylene diglycidylaniline, to form a crosslinked network or matrix. This crosslinking step is necessary to prevent dissolution of the matrix. The materials described by Sawan generally require a curing step, generally in the range of 80° to 120° C., which is unsuitable for many substrates, particularly human skin. Furthermore, the preferred organic matrix polymer (polyhexamethylene biguanide) is known to be toxic to human cells in high concentrations (see U.S. Pat. No. 6,369,289 B1). The use of silver as an antimicrobial agent also incurs some undesirable effects. One disadvantage to this approach is that certain bacteria have been able to develop resistance to silver. (Silver S., “Bacterial silver resistance: molecular biology and uses and misuses of silver compounds.” FEMS Microbiology Reviews, 2003; 27:341-353). Another disadvantage to this approach is that diffusing silver may be able to enter the wound and may potentially stain the skin. An additional disadvantage of silver is the high cost of the raw material. Similar approaches are described in U.S. Pat. Nos. 6,180,584; 6,126,931; 6,030,632; 5,869,073, 5,849,311; and 5,817,325.
Brown (EP 0136900) describes a nonwoven fabric with antimicrobial properties for use as a surgical drape. This is produced by treatment of a rayon or woodpulp material with a binder and polyhexamethylene biguanide (PHMB). Because the applied PHMB is extractable by aqueous fluids, the amount of PHMB must be kept below a critical level, in order to prevent the leached PHMB from reaching toxic levels.
Payne, in U.S. Pat. No. 5,700,742, describes treatment of textile materials with combinations of PHMB and a strong acid in order to overcome problems such as discoloration, loss of antimicrobial efficacy, and undesirable changes to substrate properties that are associated with treatment of textiles using PHMB without the added strong acid.
Orr (U.S. Pat. No. 6,369,289 B1) teaches the use of PHMB in cellulosic wound dressings. On teaches that leachable PHMB can lead to adverse effects such as skin disorders, redness, tenderness and hives, but that these are avoided by utilizing a precisely-controlled amount to PHMB in the wound dressing. Non-leaching of the applied antimicrobial is not demonstrated or even suggested. On merely teaches that the amount of leachable PHMB is less than that which would cause irritation to skin or an open wound; however he provides no data to this effect, or that the materials provide useful antimicrobial efficacy at the level of PHMB used. In fact, On calculates the amount of PHMB applied to the dressing “by extraction”, which necessarily implies that the materials can be leached or extracted. The use levels of PHMB cited by On are only slightly below those claimed by Brown (EP 0136900).
The method of Orr (U.S. Pat. No. 6,369,289 B1) is used to produce a commercial antimicrobial wound dressing known as “Kerlix-AMD”, which contains 0.2% PHMB. The Kerlix-AMD dressing is known to show a distinct zone-of-inhibition (ZOI) in a Kirby-Bauer test (see Kerlix-AMD product brochure available at (http://www.kendallhq.com/catalog/brochures/KerlixSS.pdf). A measurable ZOI is a definite indicator of leachable antimicrobial activity. The antimicrobial effectiveness of Kerlix-AMD has been described “Effectiveness of a New Antimicrobial Gauze Dressing as a Bacterial Barrier” A. M. Reitsma, et al., University of Virginia Health System, Charlottesville, Va. This study makes no mention of non-leachable properties.
PHMB has been studied as an antimicrobial treatment for cotton fabric (“Testing the Efficacy of polyhexamethylene Biguanide as an Antimicrobial Treatment for Cotton Fabric,” Michelle Wallace, AATCC Review, p 18-20, November, 2001). Antimicrobial efficacy was maintained after laundering; however, no discussion of leaching is given; however, the data does show that antimicrobial efficacy diminishes with repeated laundering cycles. Presumably, this is due to loss of PHMB from the substrate (leaching).
Consequently, there exists a need for a method that can attach an effective and non-leaching amount of antimicrobial agent to a variety of substrate materials in a convenient, reliable, and cost-effective manner. The inadequacies of existing approaches are overcome with the present inventive method wherein an improved method of non-leachably attaching antimicrobial agents to a variety of substrate materials is provided.